【作者】 李柏生；

【导师】 徐顺清；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2006， 硕士

【摘要】 纳米金(gold nanoparticle)是指直径在1—100nm之间的微小金颗粒。在过去40多年来,纳米金颗粒已经被广泛应用于免疫细胞化学以及生物标记等生物技术中。通过在纳米金颗粒表面标记上特定的寡核苷酸探针以及对纳米金颗粒表面特性的处理,开发出了一系列新的生物检测系统。利用纳米金颗粒的表面特性并结合灵敏的表面读数设备建立的Scanometric分析方法,可以检测到样本中痕量的核酸分子而不需要PCR的扩增。利用表面标记有barcode DNA和特异性配体的纳米金颗粒特异性识别相应的生物分子,并通过磁珠或者其它方式洗脱未结合的纳米颗粒探针,最后通过检测纳米颗粒上barcode DNA的量来反映目标分子的量,这就是“生物条形码”技术,已在多种痕量蛋白和核酸分子的超敏检测中应用。二恶英类化合物(Dioxin-Like Chemicals, DLCs)是包括多氯代二苯并二恶英(polychlorinated dibenzo-p-dioxins, PCDDs),多氯代二苯并呋喃(polychlorinated dibenzofurans, PCDFs)和共平面多氯联苯(polychlorinated biphenyls, PCBs)等一大类化合物的总称。DLCs毒性高,在环境中分布广泛,且不容易降解,具有生物富集和生物放大等特性,对人类健康和生态环境构成严重的危害,急需加强对其的监测。高分辨气相色谱-高分辨质谱联用检测(high resolution chromatography combined with high-resolution mass spectrometry, HRGC/HRMS)被认为是二恶英类化合物定量检测的标准方法。但该方法费时且检测费用昂贵,因此,对于能够快速、经济地检测DLCs的存在,并能准确地反映DLCs总体机体效应的生物检测方法就显得十分必要了。本研究首先建立了一个基于纳米金技术的痕量核酸分子定量检测方法,能够检测到样本中痕量的目的核酸分子;然后在此方法的基础上,引入纳米金颗粒标记的含二恶英反应元件(DRE)核心序列的双链核酸探针,根据DLCs生物学作用机制,TCDD体外诱导芳香烃受体(aryl hydrocarbon receptor,AhR)产生变构,形成TCDD-AhR-DRE复合物,在Arnt单克隆抗体的特异性捕获下,该复合物被固定于固相载体上,最后通过纳米金Scanometric检测方法建立了一种新的DLCs生物检测技术。第一部分:纳米金技术检测痕量核酸分子目的:本部分旨在建立一种新的基于纳米金技术的痕量核酸分子定量检测方法。方法:纳米金核酸探针,生物素捕获探针和目的序列在一定的离子浓度和温度条件下孵育一定时间后,杂交形成一个一端标记有纳米金颗粒,一端标记有生物素的双链DNA。该杂交产物随后被转移至亲和素包被的酶标板中,通过生物素-亲和素系统固定于酶标板中。分别用1×PBS和2×PBN溶液洗涤去除游离的纳米金探针后,用银染增强液处理,银染增强液中的银离子以纳米金颗粒为核心聚集在纳米金颗粒的表面,在还原剂的作用下还原成银原子,使得纳米金的信号得到放大。通过酶标仪在630nm波长处进行检测,可得到相应的溶液吸光度值(Optical Density,OD值)。溶液OD值变化的快慢程度与纳米金颗粒量的多少相关,而纳米金颗粒的量与目的DNA片段的浓度相关,据此可以建立一个目的DNA的浓度与溶液OD值的剂量-效应关系曲线。结果:(Ⅰ)在一定的反应时间点,溶液的OD值与溶液中目的序列的浓度相关。在整个反应过程中,目的序列的浓度越高,溶液OD值的变化越明显,而在没有目的序列的阴性对照中,溶液的OD值无明显变化。(Ⅱ)定量研究可以得到目的序列浓度-效应关系曲线,在一定范围内(10-16~10-12 mol/l),在相同的反应时间点,目的序列的浓度与溶液的OD值之间呈高度的线性相关,R2=0.9713。利用这种方法能够检测到的单链目的序列的最低浓度是100aM。(Ⅲ)对于含目的序列的双链DNA,其检测过程类似于单链检测过程,而且同样可以得到类似于单链目的DNA的浓度-效应关系曲线。由于双链DNA的特殊性,该方法对于双链DNA序列能够检测到的最低浓度是10fM。结论:核酸分子的纳米金检测法不仅灵敏特异,而且直观快速,仪器设备方便易操作,是一种灵敏实用的痕量核酸分子分析方法。第二部分:纳米金技术检测痕量二恶英类化合物目的:本部分在于探索建立一种新的基于纳米金技术的TCDD定量检测方法。方法:2,3,7,8-TCDD与含有Ah受体和Arnt蛋白的SD大鼠肝细胞溶质在20℃水浴环境中孵育一定时间后,激活Ah受体,形成2,3,7,8-TCDD-AhR-Arnt复合物。复合物中的Ah受体构像发生改变,暴露出与纳米金标记的双链DRE探针结合位点,形成2,3,7,8-TCDD-AhR-Arnt-DRE复合物。在Arnt单克隆抗体的特异性捕获下,该复合物固定于酶标板上。用1×PBS溶液和2×PBN溶液洗涤去除酶标板中的游离探针后,用银染增强液处理,并在630nm波长处实时检测溶液的OD值。溶液OD值变化的快慢程度与纳米金颗粒量的多少相关,而纳米金颗粒的量与TCDD的浓度相关。根据TCDD浓度与OD值之间的关系,绘制出相应的时间-效应曲线和剂量-效应关系曲线。结果:(Ⅰ)在一定的反应时间点,溶液的OD值与TCDD的浓度相关。在整个反应过程,TCDD浓度越高,溶液OD值的变化就越明显,而在没有TCDD的阴性对照中,溶液OD值则无明显变化。(Ⅱ)定量研究可以得到TCDD剂量-效应关系曲线,在一定的浓度范围内(10-14～10-8 mol/l),在一定的反应时间点,溶液的OD值与TCDD的浓度之间呈高度的线性相关,R2=0.9731。利用这种方法能够检测到的标准的2,3,7,8-TCDD的最低浓度是10fM。(Ⅲ)与其他的各种生物检测方法一样,纳米金技术检测二恶英方法评价的是TCDD的毒性当量(TEQs)。该方法的检测限比较低,比其他生物方法低100倍以上,且重复性好。结论:纳米金技术检测TCDD具有灵敏、快速、经济、简便的特点,是一种新颖而实用的TCDD的快速筛选的方法。本研究将纳米生物检测技术与二恶英类化合物的分析结合起来,利用纳米生物检测技术的快速灵敏和简便的特点,以期建立一种新的二恶英类化合物的快速筛选方法。与现有的其他生物检测方法相比,该方法具有很高的灵敏性、特异性与稳定性,同时它又具备简便、快速和经济的特点,特别适用于进行二恶英样本的大规模快速筛选。更多还原

【Abstract】 Gold nanoparticles are these particles with diameters between 1 and 100 nanometers. Gold nanoparticles have been used in biotechnology over the last 4 decades as immunocytochemical probes as well as biological tags. By functionalizing particles with oligonucleotides and tailoring their surface properties, there has developed a series of new and practical biodetection systems. Combined with the surface-based readouts and the property of the nanoparticles, the scanometric assay can detect the trace level of the nucleotide target without the enzyme amplification. The“bio-barcode”assay, which quantified the target molecule by amplifying the barcode DNA labeled on the surface of the nanoparticles, has paved its way to the trace level of the protein and nucleotide molecule detection field.Dioxin-like chemicals (DLCs), including polychlorinated dibenzo-p-dioxins (PCDDs), biphenyls (PCBs) and dibenzofurans (PCDFs), represent a large group of compounds. They are ubiquitous environmental pollutants which resistance to biological and chemical degradation. They are the chemicals with high bioaccumulation and biomagnifying in the food chain. It is necessary to strengthen the monitoring of DLCs and the High-resolution gas chromatography- High-resolution mass spectrometry (HRGC/HRMS) is considered as the golden standard for the identification and quantitation of DLCs. For the procedures are very costly and time-consuming, a variety of rapid and inexpensive screening bioassays capable of detecting and estimating the relative potency of complex mixtures of DLCs have been developed. Here we developed a new method for trace level of nucleotide target molecule detecting and quantifying based on the gold nanoparticle at first. This method can detect the nucleotide molecule at trace level in the sample. Then we bring the double-strand nucleotide probe including the core sequence of the Dioxin-Response-Element. We obtained the complex of TCDD-AhR-Arnt-DRE after the TCDD induction mechanism in vitro. After the complex was especially immobilized to the solid phase carrier by the anti- Arnt body, we obtained the amplified signal of the gold nanoparticle through the Silver Enhancement Solution and developed a new bio-analytic technology for DLCs detecting.Part one: Development of a novel nucleotide quantification method by the gold nanoparticleObjection: To develop a novel trace level of nucleotide target molecular quantification method based on the gold nanoparticle technology. Method: Gold nanopaticle probe, biotin-labeled probe and the target will form hybridization product with gold nanoparticle at the end and biotin at the other end of the double-strand product after incubation in the especial ion and temperature conditions for a time. Then the hybridization will be immobilized to the microplate through the avidin-biotin system. The Silver Enhancement Solution will be introduced after the microplate was washed by 1×PBS and 2×PBN to remove the free gold nanoparticle probes. The silver ions will be deoxidized to silver metal around the gold nanoparticles and the signal will be amplified. The signal was record by the Microplate Reader at 630nm as the OD value of the solution. Result: (Ⅰ) During the procedure, the color change of the solution was obviously according to the concentration of the target, while not observed for target-free groups. At a point time, the concentration of the target is higher the OD value is higher. (Ⅱ) In the range of 10-16 mol/l to 10-12 mol/l, the OD value of the solution induce by the target has concentration dependence at the same reaction time. The dose-response relationship curve about the target nuceotide concentration against the OD Value was set up and the R2=0.9713. We can detect the lowest concentration of 100aM for the single-strand target by this method. (Ⅲ) For the double-strand DNA include the target sequence, the same dose-response relationship curve can be obtain as the single-strand nucleotide. But we can only get the lowest concentration of 10fM for double-strand for the property of the double-strand. Conclusion: The gold nanoparticle technology using in the nucleotide quantification is a sensitive, rapid, simple and cost-effective method, and it can detect the even trace level of the target nucleotide specifically in the sample.Part two: Ultrasensitive colorimetric method to identify and quantify 2,3,7,8- TCDD by the gold nanoparticleObjection: To develop a ultrasensitive colorimetric method to identify and quantify 2,3,7,8- TCDD by the gold nanoparticles. Method: SD rat hepatic cytosol containing AhR and relative protein was prepared by centrifugation and was incubated with TCDD to transform the AhR. The mixture was incubated with the DRE-gold nanoparticle probe containing one DRE site to form the TCDD-AhR-Arnt-DRE complex. The complex was then immoblized on the surface of the Micro plate by the Arnt-antibody embedded previous. The unbinding probes in the mixture were then washed using 1×PBS and then 2×PBN solution. At last, it was treated with Silver Enhancement solution and read by Microplate Reader at 630nm. Results: (Ⅰ) The color change of the solution was obviously according to the concentration of TCDD, while not observed for DMSO-treated groups. At a point time, the concentration is higher the OD value is higher. (Ⅱ) In the range of 10-14 mol/l to 10-8 mol/l, the OD value of the solution induced by TCDD has dose dependence at the same reaction time. The dose-response relationship curve about TCDD concentration against the OD Value was set up and the R2=0.9731. We can detect the lowest concentration of 10fM for TCDD by this method. (Ⅲ) Just as the other bio-analytical assay, the method developed here evaluated the TEQs value of the TCDD. The method reported here was 100 times more sensitive than the other bio-analytical method. Conclusion: the gold nanoparticle technology is a sensitive, rapid, simple and cost-effective method to screen the TCDD.Here we employed the gold nanoparticle technology to the TCDD identification and quantitation. We expect to develop a useful fast screening method for dioxin-like chemicals with the sensitive and simple properties of the gold nanoparticle technology. It can be used to prescreen DLCs in environmental samples due to its consistency with the other methods, with extremely low cost. It can either evaluate the biological and toxicological effects of dioxin or be carried out easily in the field.更多还原